Mass spectrometry-based strategy for determining product-related variants of a biologic

ABSTRACT

The present invention relates to the field of protein characterization, and in particular to methods for identifying critical quality attributes of therapeutic proteins expressed in host cells by implementing a workflow including using a competitive binding assay with insufficient antigen followed by SCX-MS.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalPatent Application No. 63/221,436, filed Jul. 13, 2021 which is hereinincorporated by reference.

FIELD

The invention generally pertains to methods for determiningproduct-related variants critical for maintaining the structure andfunction of a biologic using a competitive binding-mass spectrometryworkflow.

BACKGROUND

Biologics have emerged as important drugs for the treatment of cancer,autoimmune disease, infection and cardiometabolic disorders, and theyrepresent one of the fastest growing product segments of thepharmaceutical industry. Biologics must meet very high standards ofpurity. Thus, it can be important to monitor impurities at differentstages of drug development, production, storage and handling. It isoften difficult to fully evaluate the impact of the large number ofquality attributes that may be related to safety and efficacy. Theeffects of manufacturing process parameters and material attributes onproduct quality variations are also difficult to fully characterize.

For robust manufacturing operations, it is important that an integratedcontrol strategy is developed and improved over time based on systematicprocess characterization along with implementation of appropriate riskassessment and mitigation throughout the product lifecycle. Thus, thereis a need for a quality by design standard. The U.N.'s World HealthOrganization recommends quality by design as a standard because it isharder (and practically impossible) to implement effective qualitycontrols solely by testing a product after the fact. Critical qualityattributes (CQAs) serve as the benchmarks that most quality by designimplementations revolve around. A CQA is a physical, chemical,biological, or microbiological property or characteristic that should bewithin an appropriate limit, range, or distribution to ensure thedesired product quality. CQAs are generally associated with the drugsubstance, excipients, intermediates (in-process materials), and drugproduct”. For biologics, CQAs can be product or process relatedimpurities. Product related impurities can include size variants(aggregates or fragments), variants with post-translationalmodifications or charge variants. Process related impurities are aninherent part of the process, such as the host cells' DNA or host cellproteins (HCPs), leachables (such as protein A), and viruses. Thepresence of these impurities in the final drug product can affectproduct purity, product efficacy and stability.

Identifying CQAs for biologics can, therefore, be a complicated process.Currently, liquid chromatography-tandem mass spectrometry (LC-MS/MS),electrospray ionization-mass spectrometry (ESI-MS), fractionation, orvariant identification can be used for physicochemical characterizationof intact or digested biologics. Activity characterization can beconducted using ELISA-based bioassays, cell-based bioassays, or surfaceplasmon resonance (SPR) or biolayer interferometry (BLI) for bindingactivities. For these methods, product-related CQAs need to be enrichedor isolated first and then evaluated individually or based uponexperience or prior knowledge. Such an approach to a workflow can resultin low throughput.

Thus, there is a long felt need in the art for an efficient method fordetermining such quality control attributes.

SUMMARY

Exemplary embodiments disclosed herein satisfy the aforementioneddemands by providing methods for identifying product-related CQAs byenriching them.

This disclosure provides for characterizing at least one product-relatedvariant, said method comprising obtaining a sample including a proteinof interest and at least one product-related variant of said protein ofinterest; contacting said sample to a competitive binding conditionincluding an insufficient target immobilized on beads; washing saidbeads to collect a flow-through; subjecting said flow-through to liquidchromatography-mass spectrometry analysis to separate said protein ofinterest and said at least one product-related variant; and comparingthe abundance of said at least one product-related variant to anabundance of said at least one product-related variant obtained from aliquid chromatography-mass spectrometry analysis of a control sampleprior to contacting said sample to said competitive binding condition tocharacterize said at least one product-related variant.

In one aspect of this embodiment, the target is an antigen against whichthe protein of interest is directed.

In one aspect of this embodiment, the binding condition provides aninsufficient target immobilized on beads. In the same or another aspectof this embodiment, the at least one product-related variant hascompromised binding with said insufficient target.

In one aspect of this embodiment, the liquid chromatography iscation-exchange chromatography. In a specific aspect of this embodiment,the liquid chromatography is a strong cation-exchange chromatography.

In one aspect of this embodiment, the mass spectrometer is anelectrospray ionization mass spectrometer. In a specific aspect of thisembodiment, the mass spectrometer is a nano-electrospray ionization massspectrometer

In one aspect of this embodiment, said beads are magnetic. In anotheraspect of this embodiment, said beads are non-magnetic. In a furtheraspect, said beads are agarose beads. In yet another aspect, said beadsare capable of being coated with a peptide or a protein.

In one aspect of this embodiment, wherein said flow-through is enrichedfor said at least one product-related variant.

In the same or another aspect of this embodiment, said flow-through iscollected by performing centrifugation.

In one aspect of this embodiment, said target is biotinylated beforeimmobilizing on said beads. In the same or other aspects of thisembodiment, said beads are coated with streptavidin resin. In a specificaspect of this embodiment, said beads are non-magnetic. In anotherspecific aspect, said beads are magnetic.

In one aspect of this embodiment, said insufficient target is such thatthe amount of said target allows binding of about 30% to about 80% ofthe protein of interest.

In another aspect of this embodiment, said sample is incubated for aboutan hour prior to washing. In the same or other aspects of thisembodiment, said sample is incubated at room temperature prior towashing.

In one aspect of this embodiment, the method is capable of identifyingmore than one product-related variant. In a specific aspect, saidproduct-related variant comprises a size-variant. In a specific aspect,said size-variant is a fragmentation variant of said protein ofinterest. In a specific aspect, said size-variant is an aggregationvariant of said protein of interest.

In one aspect of the embodiment, said product-related variant comprisesa charge-variant of said protein of interest. In a specific aspect, saidproduct-related variant comprises a post translationallymodified-variant of said protein of interest.

In one aspect of this embodiment, said product-related variant isclassified as a critical quality attribute if said abundance of said atleast product-related variant is significantly more than said abundanceof said at least product-related variant in the sample prior tocontacting said sample to said competitive binding condition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of possible different product-relatedvariants of an antibody including size variants, charge variants, andpost-translational modifications (PTMs).

FIG. 2 is a representation of methods routinely used to determine ormonitor CQAs during protein drug development.

FIG. 3A shows a method for identifying at least one product-relatedvariant according to an exemplary embodiment.

FIG. 3B shows a method for identifying at least one product-relatedvariant according to an exemplary embodiment.

FIG. 4 shows a method design and workflow for a method for identifyingat least one product-related variant according to an exemplaryembodiment.

FIG. 5 shows a method design and workflow to determine the antigen toantibody ratio according to an exemplary embodiment.

FIG. 6 shows a titration curve obtained to determine the antigen toantibody ratio according to an exemplary embodiment.

FIG. 7 shows a chromatogram of a sample not enriched for product-relatedvariants of mAb1 according to an exemplary embodiment.

FIG. 8 shows comparison of chromatograms of a sample enriched forproduct-related variants of mAb1 with reduced binding affinity accordingto an exemplary embodiment and the control experiment.

FIG. 9 shows comparison of extracted ion chromatograms (XICs) ofdifferent product-related variants of mAb1 enriched for product-relatedvariants with reduced binding affinity according to an exemplaryembodiment to the control experiment.

FIG. 10 shows a chart of relative percentages of product-relatedvariants of mAb1 identified using a method according to an exemplaryembodiment and control experiment.

FIG. 11 shows the structure of bsAb1.

FIG. 12 shows a comparison of XICs of a sample enriched forproduct-related variants of mAb2 with reduced binding affinity accordingto an exemplary embodiment and the control experiment.

FIG. 13 shows a chart of relative percentages of the deamidation variantof bsAb1 identified using a method according to an exemplary embodimentand control experiment.

FIG. 14 shows a chart of relative percentages of product-relatedvariants of bsAb1 identified using a method according to an exemplaryembodiment and control experiment.

DETAILED DESCRIPTION

Identification and quantification of product-related variants inbiologic products can be very important during the production anddevelopment of a product. The identification of such variants can beimperative into developing a safe and effective product. Hence, a robustmethod and/or workflow to characterize CQAs can be beneficial.

The Annex to ICH Q8 defines CQAs as physical, chemical, biological ormicrobiological properties or characteristics that should be within anappropriate limit, range or distribution to ensure the desired productquality, safety/immunogenicity, efficacy andpharmacodynamics/pharmacokinetics. (US Food and Drug Administration.Guidance for industry: Q8(R2) pharmaceutical development.www.fda.gov/media/71535/download). Thus, CQAs must be within anappropriate limit, range or distribution to ensure the desired productquality, safety and efficacy. For example, for monoclonal antibodytherapeutics that rely on fraction crystalizable (Fc)-mediated effectorfunction for their clinical activity, the terminal sugars of Fc glycanshave been shown to be critical for safety or efficacy. Such CQAs includeproduct-related variants, such as size and charge variants, which canimpact binding of the protein of interest.

FIG. 1 shows a non-limiting example of variants that can affect thecritical quality attribute of a protein. In case of the antibodyrepresented in FIG. 1 , the product-related impurities can be sizevariants like fragmentation products (LMW) and aggregation products(HMW). Other product-related impurities can be charge variants formeddue to N-term blocking, disulfide bond formation, C-term clipping, Fcglycan microheterogeneity, or post-translational modifications. Thesecan cause decrease binding of the protein of interest and need to bemonitored at various parts of the manufacturing and delivery process.

One of the conventional methods includes use of strong cation exchangechromatography (SCX). One such workflow is shown in FIG. 2 . Thisincludes performing separation of the protein of interest and itsvariants by SCX followed by conducting a binding assay of the protein ofinterest and its variant to identify if the variant has compromised,i.e., reduced binding affinity compared to the protein of interest.

Considering the limitations of existing methods, effective and efficientmethods for identification and quantification of dimer species wasdeveloped.

Unless described otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing, particular methods and materials arenow described. All publications mentioned are hereby incorporated byreference.

The term “a” should be understood to mean “at least one”; and the terms“about” and “approximately” should be understood to permit standardvariation as would be understood by those of ordinary skill in the art;and where ranges are provided, endpoints are included.

In some exemplary embodiments, the disclosure provides a methodidentifying at least one product-related variant in a sample comprisinga protein of interest.

As used herein, the term “protein” or “protein of interest” includes anyamino acid polymer having covalently linked amide bonds. Proteinscomprise one or more amino acid polymer chains, generally known in theart as “polypeptides”. “Polypeptide” refers to a polymer composed ofamino acid residues, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof linked via peptidebonds, related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. “Synthetic peptides orpolypeptides' refers to a non-naturally occurring peptide orpolypeptide. Synthetic peptides or polypeptides can be synthesized, forexample, using an automated polypeptide synthesizer. Various solid phasepeptide synthesis methods are known. A protein may contain one ormultiple polypeptides to form a single functioning biomolecule. Aprotein can include any of biotherapeutic proteins, recombinant proteinsused in research or therapy, trap proteins and other chimeric receptorFc-fusion proteins, chimeric proteins, antibodies, monoclonalantibodies, polyclonal antibodies, human antibodies, and bispecificantibodies. In another exemplary aspect, a protein can include antibodyfragments, nanobodies, recombinant antibody chimeras, cytokines,chemokines, peptide hormones, and the like. Proteins may be producedusing recombinant cell-based production systems, such as the insectbacculovirus system, yeast systems (e.g., Pichia sp.), mammalian systems(e.g., CHO cells and CHO derivatives like CHO-K1 cells). For a reviewdiscussing biotherapeutic proteins and their production, see Ghaderi etal., “Production platforms for biotherapeutic glycoproteins. Occurrence,impact, and challenges of non-human sialylation,” (BIOTECHNOL. GENET.ENG. REV. 147-175 (2012)). In some exemplary embodiments, proteinscomprise modifications, adducts, and other covalently linked moieties.Those modifications, adducts and moieties include for example avidin,streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose,neuraminic acid, N-acetylglucosamine, fucose, mannose, and othermonosaccharides), PEG, polyhistidine, FLAGtag, maltose binding protein(MBP), chitin binding protein (CBP), glutathione-S-transferase (GST)myc-epitope, fluorescent labels and other dyes, and the like. Proteinscan be classified on the basis of compositions and solubility and canthus include simple proteins, such as, globular proteins and fibrousproteins; conjugated proteins, such as nucleoproteins, glycoproteins,mucoproteins, chromoproteins, phosphoproteins, metalloproteins, andlipoproteins; and derived proteins, such as primary derived proteins andsecondary derived proteins.

In some exemplary embodiments, the protein can be an antibody, abispecific antibody, a multispecific antibody, antibody fragment,monoclonal antibody, or an Fc fusion protein.

The term “antibody,” as used herein includes immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds, as well as multimersthereof (e.g., IgM). Each heavy chain comprises a heavy chain variableregion (abbreviated herein as HCVR or V_(H)) and a heavy chain constantregion. The heavy chain constant region comprises three domains, C_(H)1,C_(H2) and C_(H)3. Each light chain comprises a light chain variableregion (abbreviated herein as LCVR or V_(L)) and a light chain constantregion. The light chain constant region comprises one domain (Cu). TheV_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different exemplaryembodiments, the FRs of the anti-big-ET-1 antibody (or antigen-bindingportion thereof) may be identical to the human germline sequences, ormay be naturally or artificially modified. An amino acid consensussequence may be defined based on a side-by-side analysis of two or moreCDRs. The term “antibody,” as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

As used herein, an “antibody fragment” includes a portion of an intactantibody, such as, for example, the antigen-binding or variable regionof an antibody. Examples of antibody fragments include, but are notlimited to, a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a Fcfragment, a scFv fragment, a Fv fragment, a dsFv diabody, a dAbfragment, a Fd′ fragment, a Fd fragment, and an isolated complementaritydetermining region (CDR) region, as well as triabodies, tetrabodies,linear antibodies, single-chain antibody molecules, and multi specificantibodies formed from antibody fragments. Fv fragments are thecombination of the variable regions of the immunoglobulin heavy andlight chains, and ScFv proteins are recombinant single chain polypeptidemolecules in which immunoglobulin light and heavy chain variable regionsare connected by a peptide linker. An antibody fragment may be producedby various means. For example, an antibody fragment may be enzymaticallyor chemically produced by fragmentation of an intact antibody and/or itmay be recombinantly produced from a gene encoding the partial antibodysequence. Alternatively or additionally, an antibody fragment may bewholly or partially synthetically produced. An antibody fragment mayoptionally comprise a single chain antibody fragment. Alternatively oradditionally, an antibody fragment may comprise multiple chains that arelinked together, for example, by disulfide linkages. An antibodyfragment may optionally comprise a multi-molecular complex.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. A monoclonal antibodycan be derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, by any means available or known in the art.Monoclonal antibodies useful with the present disclosure can be preparedusing a wide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof.

The term “Fc fusion proteins” as used herein includes part or all of twoor more proteins, one of which is an Fc portion of an immunoglobulinmolecule, that are not fused in their natural state. Preparation offusion proteins comprising certain heterologous polypeptides fused tovarious portions of antibody-derived polypeptides (including the Fcdomain) has been described, e.g., by Ashkenazi et al., Proc. Natl. Acad.ScL USA 88: 10535, 1991; Byrn et al., Nature 344:677, 1990; andHollenbaugh et al., “Construction of Immunoglobulin Fusion Proteins”, inCurrent Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992.“Receptor Fc fusion proteins” comprise one or more of one or moreextracellular domain(s) of a receptor coupled to an Fc moiety, which insome embodiments comprises a hinge region followed by a CH2 and CH3domain of an immunoglobulin. In some embodiments, the Fc-fusion proteincontains two or more distinct receptor chains that bind to a single ormore than one ligand(s). For example, an Fc-fusion protein is a trap,such as for example an IL-1 trap (e.g., Rilonacept, which contains theIL-1 RAcP ligand binding region fused to the IL-1R1 extracellular regionfused to Fc of hIgG1; see U.S. Pat. No. 6,927,004, which is hereinincorporated by reference in its entirety), or a VEGF Trap (e.g.,Aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1fused to the Ig domain 3 of the VEGF receptor Flk1 fused to Fc of hIgG1;e.g., SEQ ID NO:1; see U.S. Pat. Nos. 7,087,411 and 7,279,159, which areherein incorporated by reference in their entirety).

As used herein, the term “target” refers to any molecule that mayspecifically interact with a therapeutic protein in order to achieve apharmacological effect. For example, the target of an antibody may be anantigen against which it is directed; the target of a ligand may be areceptor to which it preferentially binds, and vice versa; the target ofan enzyme may be a substrate to which it preferentially binds; and soforth. A single therapeutic protein may have more than one target. Avariety of targets are suitable for use in the method of the invention,according to the specific application. A target may, for example, bepresent on a cell surface, may be soluble, may be cytosolic, or may beimmobilized on a solid surface. A target may be recombinant protein. Insome exemplary embodiments, the target may be an antigen.

As used herein, the term “impurity” can include any undesirable proteinpresent in the protein biopharmaceutical product. Impurity can includeprocess and product-related impurities. The impurity can further be ofknown structure, partially characterized, or unidentified.

Process-related impurities can be derived from the manufacturing processand can include the three major categories: cell substrate-derived, cellculture-derived and downstream derived. Cell substrate-derivedimpurities include, but are not limited to, proteins derived from thehost organism and nucleic acid (host cell genomic, vector, or totalDNA). Cell culture-derived impurities include, but are not limited to,inducers, antibiotics, serum, and other media components.Downstream-derived impurities include, but are not limited to, enzymes,chemical and biochemical processing reagents (e.g., cyanogen bromide,guanidine, oxidizing and reducing agents), inorganic salts (e.g., heavymetals, arsenic, nonmetallic ion), solvents, carriers, ligands (e.g.,monoclonal antibodies), and other leachables.

Product-related impurities (e.g., precursors, certain degradationproducts) can be molecular variants arising during manufacture and/orstorage that do not have properties comparable to those of the desiredproduct with respect to activity, efficacy, and safety. Such variantsmay need considerable effort in isolation and characterization in orderto identify the type of modification(s). Product-related impurities caninclude truncated forms, modified forms, and aggregates. Truncated formsare formed by hydrolytic enzymes or chemicals which catalyze thecleavage of peptide bonds. Modified forms include, but are not limitedto, deamidated, isomerized, mismatched S—S linked, oxidized, or alteredconjugated forms (e.g., glycosylation, phosphorylation). Modified formscan also include any post-translationally modified form. Aggregatesinclude dimers and higher multiples of the desired product. (Q6BSpecifications: Test Procedures and Acceptance Criteria forBiotechnological/Biological Products, ICH August 1999, U.S. Dept. ofHealth and Humans Services).

Some product-related impurities or product-related protein variants havecompromised binding affinity. Compromised binding affinity, here,includes a reduced binding affinity to the target of the protein ofinterest in the body or an antigen designed for the protein of interest.The compromised binding affinity can be any affinity which is less thanthe affinity of the protein of interest towards the target of theprotein of interest in the body or an antigen designed for the proteinof interest.

As used herein, the general term “post-translational modifications” or“PTMs” refers to covalent modifications that polypeptides undergo,either during (co-translational modification) or after(post-translational modification) their ribosomal synthesis. PTMs aregenerally introduced by specific enzymes or enzyme pathways. Many occurat the site of a specific characteristic protein sequence (signaturesequence) within the protein backbone. Several hundred PTMs have beenrecorded, and these modifications invariably influence some aspect of aprotein's structure or function (Walsh, G. “Proteins” (2014) secondedition, published by Wiley and Sons, Ltd., ISBN: 9780470669853). Thevarious post-translational modifications include, but are not limitedto, cleavage, N-terminal extensions, protein degradation, acylation ofthe N-terminus, biotinylation (acylation of lysine residues with abiotin), amidation of the C-terminal, glycosylation, iodination,covalent attachment of prosthetic groups, acetylation (the addition ofan acetyl group, usually at the N-terminus of the protein), alkylation(the addition of an alkyl group (e.g. methyl, ethyl, propyl) usually atlysine or arginine residues), methylation, adenylation,ADP-ribosylation, covalent cross links within, or between, polypeptidechains, sulfonation, prenylation, Vitamin C dependent modifications(proline and lysine hydroxylations and carboxy terminal amidation),Vitamin K dependent modification wherein Vitamin K is a cofactor in thecarboxylation of glutamic acid residues resulting in the formation of aγ-carboxyglutamate (a glu residue), glutamylation (covalent linkage ofglutamic acid residues), glycylation (covalent linkage glycineresidues), glycosylation (addition of a glycosyl group to eitherasparagine, hydroxylysine, serine, or threonine, resulting in aglycoprotein), isoprenylation (addition of an isoprenoid group such asfarnesol and geranylgeraniol), lipoylation (attachment of a lipoatefunctionality), phosphopantetheinylation (addition of a4′-phosphopantetheinyl moiety from coenzyme A, as in fatty acid,polyketide, non-ribosomal peptide and leucine biosynthesis),phosphorylation (addition of a phosphate group, usually to serine,tyrosine, threonine or histidine), and sulfation (addition of a sulfategroup, usually to a tyrosine residue). The post-translationalmodifications that change the chemical nature of amino acids include,but are not limited to, citrullination (the conversion of arginine tocitrulline by deimination), and deamidation (the conversion of glutamineto glutamic acid or asparagine to aspartic acid). The post-translationalmodifications that involve structural changes include, but are notlimited to, formation of disulfide bridges (covalent linkage of twocysteine amino acids) and proteolytic cleavage (cleavage of a protein ata peptide bond). Certain post-translational modifications involve theaddition of other proteins or peptides, such as ISGylation (covalentlinkage to the ISG15 protein (Interferon-Stimulated Gene)), SUMOylation(covalent linkage to the SUMO protein (Small Ubiquitin-relatedMOdifier)) and ubiquitination (covalent linkage to the proteinubiquitin). See European Bioinformatics Institute Protein InformationResourceSIB Swiss Institute of Bioinformatics, EUROPEAN BIOINFORMATICSINSTITUTE DRS—DROSOMYCIN PRECURSOR— Drosophila Melanogaster (FRUITFLY)—DRS GENE & PROTEIN, http://www.uniprot.org/docs/ptmlist (lastvisited Jan. 15, 2019) for a more detailed controlled vocabulary of PTMscurated by UniProt.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas can be separated intocomponents as a result of differential distribution of the chemicalentities as they flow around or over a stationary liquid or solid phase.Non-limiting examples of chromatography include traditionalreversed-phased (RP), ion exchange (IEX), mixed mode chromatography andnormal phase chromatography (NP).

As used herein, the term “cation exchange chromatography” means achromatography method which uses a “cation exchange chromatographymaterial”. Further depending on the nature of the charged group the“cation exchange chromatography material” is referred to as e.g. in thecase of cation exchange chromatography materials with sulfonic acidgroups (S), or carboxymethyl groups (CM). Depending on the chemicalnature of the charged group the “cation exchange chromatographymaterial” can additionally be classified as strong or weak ion exchangechromatography material, depending on the strength of the covalentlybound charged substituent. For example, strong cation exchangechromatography materials have a sulfonic acid group as chromatographicfunctional group.

For example, “cation exchange chromatography materials”, for example,are available under different names from a multitude of companies suchas e.g. Bio-Rex, Macro-Prep CM (available from BioRad Laboratories,Hercules, Calif., USA), weak cation exchanger WCX 2 (available fromCiphergen, Fremont, Calif., USA), Dowex MAC-3 (available from Dowchemical company, Midland, Mich., USA), Mustang C (available from PallCorporation, East Hills, N.Y., USA), Cellulose CM-23, CM-32, CM-52,hyper-D, and partisphere (available from Whatman plc, Brentford, UK),Amberlite IRC 76, IRC 747, IRC 748, GT 73 (available from TosohBioscience GmbH, Stuttgart, Germany), CM 1500, CM 3000 (available fromBioChrom Labs, Terre Haute, Ind., USA), and CM-Sepharose Fast Flow(available from GE Healthcare, Life Sciences, Germany). In addition,commercially available cation exchange resins further includecarboxymethyl-cellulose, Bakerbond ABX, sulphopropyl (SP) immobilized onagarose (e.g. SP-Sepharose Fast Flow or SP-Sepharose High Performance,available from GE Healthcare—Amersham Biosciences Europe GmbH, Freiburg,Germany) and sulphonyl immobilized on agarose (e.g. S-Sepharose FastFlow available from GE Healthcare, Life Sciences, Germany).

The “cation exchange chromatography materials” include mixed-modechromatography materials performing a combination of ion exchange andhydrophobic interaction technologies (e.g., Capto adhere, Capto MMC, MEPHyperCell, Eshmuno HCX, etc.), mixed-mode chromatography material sperforming a combination of anion exchange and cation exchangetechnologies (e.g., hydroxyapatite, ceramic hydroxyapatite, etc.), andthe like. Cation exchange chromatography materials that may be used incation exchange chromatography in the present invention may include, butare not limited to, all the commercially available cation exchangechromatography materials as described above. In an example of thepresent invention YMC BioPro SP-F column was used as cation exchangechromatography material.

As used herein, the term “mass spectrometer” includes a device capableof identifying specific molecular species and measuring their accuratemasses. The term is meant to include any molecular detector into which apolypeptide or peptide may be eluted for detection and/orcharacterization. A mass spectrometer can include three major parts: theion source, the mass analyzer, and the detector. The role of the ionsource is to create gas phase ions. Analyte atoms, molecules, orclusters can be transferred into gas phase and ionized eitherconcurrently (as in electrospray ionization). The choice of ion sourcedepends heavily on the application.

In some embodiments, the mass spectrometer can be an electrospray-massspectrometer.

As used herein, the term “electrospray ionization” or “ESI” refers tothe process of spray ionization in which either cations or anions insolution are transferred to the gas phase via formation and desolvationat atmospheric pressure of a stream of highly charged droplets thatresult from applying a potential difference between the tip of theelectrospray needle containing the solution and a counter electrode.There are generally three major steps in the production of gas-phaseions from electrolyte ions in solution. These are: (a) production ofcharged droplets at the ES infusion tip; (b) shrinkage of chargeddroplets by solvent evaporation and repeated droplet disintegrationsleading to small highly charged droplets capable of producing gas-phaseions; and (c) the mechanism by which gas-phase ions are produced fromvery small and highly charged droplets. Stages (a)-(c) generally occurin the atmospheric pressure region of the apparatus.

As used herein, the term “electrospray infusion setup” refers to anelectrospray ionization system that is compatible with a massspectrometer used for mass analysis of protein. In electrosprayionization, an electrospray needle has its orifice positioned close tothe entrance orifice of a spectrometer. A sample, containing the proteinof interest, can be pumped through the syringe needle. An electricpotential between the syringe needle orifice and an orifice leading tothe mass analyzer forms a spray (“electrospray”) of the solution. Theelectrospray can be carried out at atmospheric pressure and provideshighly charged droplets of the solution. The electrospray infusion setupcan include an electrospray emitter, nebulization gas, and/or an ESIpower supply. The setup can optionally be automated to carry out sampleaspiration, sample dispensing, sample delivery, and/or for spraying thesample.

In some exemplary embodiments, the electrospray ionization massspectrometer can be a nano-electrospray ionization mass spectrometer.

The term “nanoelectrospray” or “nanospray” as used herein refers toelectrospray ionization at a very low solvent flow rate, typicallyhundreds of nanoliters per minute of sample solution or lower, oftenwithout the use of an external solvent delivery. The electrosprayinfusion setup forming a nanoelectrospray can use a staticnanoelectrospray emitter or a dynamic nanoelectrospray emitter. A staticnanoelectrospray emitter performs a continuous analysis of small sample(analyte) solution volumes over an extended period of time. A dynamicnanoelectrospray emitter uses a capillary column and a solvent deliverysystem to perform chromatographic separations on mixtures prior toanalysis by the mass spectrometer.

As used herein, the term “mass analyzer” includes a device that canseparate species, that is, atoms, molecules, or clusters, according totheir mass. Non-limiting examples of mass analyzers that could beemployed for fast protein sequencing are time-of-flight (TOF),magnetic/electric sector, quadrupole mass filter (Q), quadrupole iontrap (QIT), orbitrap, Fourier transform ion cyclotron resonance (FTICR),and also the technique of accelerator mass spectrometry (AMS).

In some exemplary embodiments, mass spectrometry can be performed undernative conditions.

As used herein, the term “native conditions” or “native MS” or “nativeESI-MS” can include a performing mass spectrometry under conditions thatpreserve no-covalent interactions in an analyte. For detailed review onnative MS, refer to the review: Elisabetta Boeri Erba & Carlo Petosa,The emerging role of native mass spectrometry in characterizing thestructure and dynamics of macromolecular complexes, 24 PROTEIN SCIENCE1176-1192 (2015). Some of the distinctions between native ESI andregular ESI are illustrated in table 1 and FIG. 1 (Hao Zhang et al.,Native mass spectrometry of photosynthetic pigment-protein complexes,587 FEBS Letters 1012-1020 (2013)).

In some exemplary embodiments, the mass spectrometer can be a tandemmass spectrometer.

As used herein, the term “tandem mass spectrometry” includes a techniquewhere structural information on sample molecules is obtained by usingmultiple stages of mass selection and mass separation. A prerequisite isthat the sample molecules can be transferred into gas phase and ionizedintact and that they can be induced to fall apart in some predictableand controllable fashion after the first mass selection step. MultistageMS/MS, or MS^(n), can be performed by first selecting and isolating aprecursor ion (MS²), fragmenting it, isolating a primary fragment ion(MS³), fragmenting it, isolating a secondary fragment (MS⁴), and so onas long as one can obtain meaningful information or the fragment ionsignal is detectable. Tandem MS have been successfully performed with awide variety of analyzer combinations. What analyzers to combine for acertain application is determined by many different factors, such assensitivity, selectivity, and speed, but also size, cost, andavailability. The two major categories of tandem MS methods aretandem-in-space and tandem-in-time, but there are also hybrids wheretandem-in-time analyzers are coupled in space or with tandem-in-spaceanalyzers. A tandem-in-space mass spectrometer comprises an ion source,a precursor ion activation device, and at least two non-trapping massanalyzers. Specific m/z separation functions can be designed so that inone section of the instrument ions are selected, dissociated in anintermediate region, and the product ions are then transmitted toanother analyzer for m/z separation and data acquisition. Intandem-in-time mass spectrometer ions produced in the ion source can betrapped, isolated, fragmented, and m/z separated in the same physicaldevice.

The peptides identified by the mass spectrometer can be used assurrogate representatives of the intact protein and theirpost-translational modifications. They can be used for proteincharacterization by correlating experimental and theoretical MS/MS data,the latter generated from possible peptides in a protein sequencedatabase. The characterization can include, but is not limited, tosequencing amino acids of the protein fragments, determining proteinsequencing, determining protein de novo sequencing, locatingpost-translational modifications, or identifying post translationalmodifications, or comparability analysis, or combinations thereof.

As used herein, the term “database” refers to a compiled collection ofprotein sequences that may possibly exist in a sample, for example inthe form of a file in a FASTA format. Relevant protein sequences may bederived from cDNA sequences of a species being studied. Public databasesthat may be used to search for relevant protein sequences includeddatabases hosted by, for example, Uniprot or Swiss-prot. Databases maybe searched using what are herein referred to as “bioinformatics tools”.Bioinformatics tools provide the capacity to search uninterpreted MS/MSspectra against all possible sequences in the database(s), and provideinterpreted (annotated) MS/MS spectra as an output. Non-limitingexamples of such tools are Mascot (www.matrixscience.com), Spectrum Mill(www.chem.agilent.com), PLGS (www.waters.com), PEAKS(www.bioinformaticssolutions.com), Proteinpilot(download.appliedbiosystems.com//proteinpilot), Phenyx(www.phenyx-ms.com), Sorcerer (www.sagenresearch.com), OMS SA(www.pubchem.ncbi.nlm.nih.gov/omssa/), X!Tandem(www.thegpm.org/TANDEM/), Protein Prospector(prospector.ucsfedu/prospector/mshome.htm), Byonic(www.proteinmetrics.com/products/byonic) or Sequest(fields.scripps.edu/sequest).

In some embodiments, the method for identifying at least oneproduct-related variant can comprise using a competitive binding assaywith insufficient antigen immobilized on a solid surface.

As used herein, the term “solid surface” can include any surface with anability to bind to an antigen. Non-limiting examples of solid surfacecan include affinity resins, beads and coated plates with an immobilizedprotein, such as, avidin, streptavidin, or NeutrAvidin.

In some embodiments, the sample comprising the protein of interest canbe digested after the competitive binding assay but prior to assessingit through SCX-MS.

In some embodiments, the sample comprising the protein of interest canbe treated by adding a reducing agent to the sample.

As used herein, the term “reducing” refers to the reduction of disulfidebridges in a protein. Non-limiting examples of the reducing agents usedto reduce the protein are dithiothreitol (DTT), ß-mercaptoethanol,Ellman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride,tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or combinationsthereof. In some specific embodiments, the treatment can further includealkylation. In some other specific exemplary embodiments, the treatmentcan include alkylation of sulfhydryl groups on a protein.

As used herein, the term “treating” or “isotopically labeling” can referto chemical labeling a protein. Non-limiting examples of methods tochemically label a protein include Isobaric tags for relative andabsolute quantitation (iTRAQ) using reagents, such as 4-plex, 6-plex,and 8-plex; reductive demethylation of amines, carbamylation of amines,¹⁸O-labeling on the C-terminus of the protein, or any amine- orsulfhydryl-group of the protein to label amines or sulfhydryl group.

As used herein, the term “digestion” refers to hydrolysis of one or morepeptide bonds of a protein. There are several approaches to carrying outdigestion of a protein in a sample using an appropriate hydrolyzingagent, for example, enzymatic digestion or non-enzymatic digestion.

As used herein, the term “hydrolyzing agent” refers to any one orcombination of a large number of different agents that can performdigestion of a protein. Non-limiting examples of hydrolyzing agents thatcan carry out enzymatic digestion include trypsin, endoproteinase Arg-C,endoproteinase Asp-N, endoproteinase Glu-C, outer membrane protease T(OmpT), immunoglobulin-degrading enzyme of Streptococcus pyogenes(IdeS), chymotrypsin, pepsin, thermolysin, papain, pronase, and proteasefrom Aspergillus Saitoi. Non-limiting examples of hydrolyzing agentsthat can carry out non-enzymatic digestion include the use of hightemperature, microwave, ultrasound, high pressure, infrared, solvents(non-limiting examples are ethanol and acetonitrile), immobilized enzymedigestion (IMER), magnetic particle immobilized enzymes, and on-chipimmobilized enzymes. For a recent review discussing the availabletechniques for protein digestion see Switazar et al., “ProteinDigestion: An Overview of the Available Techniques and RecentDevelopments” (J. Proteome Research 2013, 12, 1067-1077). One or acombination of hydrolyzing agents can cleave peptide bonds in a proteinor polypeptide, in a sequence-specific manner, generating a predictablecollection of shorter peptides.

Exemplary Embodiments

Embodiments disclosed herein provide methods for identifying at leastone product-related variant in a sample comprising a protein ofinterest.

In some exemplary embodiments, this disclosure provides a method foridentifying at least one product-related variant in a sample comprisinga protein of interest, contacting a sample including a protein ofinterest and at least one product-related variant to a competitivebinding condition, wherein a binding condition provides an insufficientantigen immobilized on beads and wherein said at least oneproduct-related variant has compromised binding with said insufficientantigen; incubating said sample with said insufficient antigen;collecting a flow-through from washing after incubating; and identifyingthe at least one product-related critical quality attributes in saidflow-through using a liquid chromatography-mass spectrometer.

In some exemplary embodiments, a product-related variant is one or moreof truncated forms, modified forms, and aggregates of the protein ofinterest.

In some exemplary embodiments, a product-related variant is deamidated,isomerized, mismatched S—S linked, oxidized, and/or altered conjugatedform (e.g., glycosylation, phosphorylation) of the protein of interest.

In some exemplary embodiments, a product-related variant is apost-translationally modified form.

In some exemplary embodiments, a product-related variant has acompromised binding affinity, wherein the compromised binding affinityis about 90% the binding affinity of the protein of interest, about 80%the binding affinity of the protein of interest, about 70% the bindingaffinity of the protein of interest, about 60% the binding affinity ofthe protein of interest, about 50% the binding affinity of the proteinof interest, about 40% the binding affinity of the protein of interest,about 30% the binding affinity of the protein of interest, about 20% thebinding affinity of the protein of interest, or is about 10% the bindingaffinity of the protein of interest.

In some exemplary embodiments, the mass spectrometer can be anano-electrospray ionization mass spectrometer.

In some exemplary embodiments, the electrospray ionization massspectrometer can be run under native conditions.

It is understood that the methods are not limited to any of theaforesaid protein, impurity, and column and that the methods foridentifying or quantifying may be conducted by any suitable means.

An exemplary embodiment is illustrated in FIGS. 3A and 3B. To a samplecomprising the protein if interest and possibly its variants, beads withimmobilized antigen can be added. The amount of beads with immobilizedantigen is such that not all the protein of interest (native mAb) andits variants can bind to it. Any variant with a reduced binding affinityto the antigen will have lower chance to bind due to the limited amountof antigen present. The flow-through (unbound fraction) can be collectedand analyzed using SCX-MS or peptide mapping. The control (i.e., thesample without the immobilized antigen binding assay step) can also beanalyzed using SCX-MS or peptide mapping. The comparative study betweenthe flow-through and control can lead to a chromatogram as illustratedin FIG. 3B. Any variant with reduced binding affinity would be moreabundant in the flow-through. On comparison of the amount of the variantas such identified, it can be seen that the relative percentage of thevariant is more in the flow-through than the control due to its reducedbinding affinity.

Such an experiment can be devised using the workflow as shown in FIG. 4and FIG. 5 .

For the present invention, the ratio between the antigen and protein ofinterest is very important. The amount of the antigen added can be suchthat about 25% to about 75% of the protein of interest can bind to theantigen. In some embodiments, the amount of the antigen added can besuch that about 50% of the protein of interest can bind to the antigen.

The consecutive labeling of method steps as provided herein with numbersand/or letters is not meant to limit the method or any embodimentsthereof to the particular indicated order.

Various publications, including patents, patent applications, publishedpatent applications, accession numbers, technical articles and scholarlyarticles are cited throughout the specification. Each of these citedreferences is herein incorporated by reference, in its entirety and forall purposes.

The disclosure will be more fully understood by reference to thefollowing Examples, which are provided to describe the disclosure ingreater detail. They are intended to illustrate examples and should notbe construed as limiting the scope of the disclosure.

EXAMPLES

Materials. Deionized water was provided by a Milli-Q integral waterpurification system installed with a MilliPak Express 20 filter(Millipore Sigma, Burlington, Mass.). mAb1, mAb2, mAb1 antigen, and mAb2antigen were generated in-house at Regeneron (Tarrytown, N.Y.).

Online nSCX-UV/MS Analysis.

Strong cation exchange chromatography was performed using a YMC BioProSP-F (YMC, Japan). For the sample separations, the mobile phases usedwere 20 mM Ammonium Acetate, pH 5.6 (Mobile phase A) and 150 mM AmmoniumAcetate pH 7.4 (Mobile phase B). A linear pH gradient was used to elutecharge variants of mAb1 with detection at 280 nm.

Prior to sample injection, the column compartment temperature was set at45° C. and a strong cation exchange column (100 mm 4.6 mm, 5 μm) (YMC,Japan) was preconditioned with mobile phase A (20 mM ammonium acetate,pH adjusted to 5.6 with 20 mM acetic acid) at a flow rate of 0.4 mL/min.Upon the injection of an aliquot (10 μg) of the protein samples, thegradient is held at 100% mobile phase A for 2 minutes followed by alinear increase to 100% mobile phase B (150 mM ammonium acetate, pH 7.4)in 16 minutes. The gradient was held at 100% mobile phase B for 4minutes and then returned to 100% mobile phase A to recondition thecolumn for 7 minutes before the next injection. Peaks at a relativeresidence time earlier or later than the main peak are identified usingan online MS.

For the mass spectrometric analysis, the resolution was set at 17,500,the capillary spray voltage was set at 1.5 kV, the in-sourcefragmentation energy was set at 100, the collision energy was set at 10,the capillary temperature was set at 350° C., the S-lens RF level wasset at 200 and the HCD trapping gas pressure was set at 3. Mass spectrawere acquired with an m/z range window between 2000 and 15000.

Data analysis. Protein Metrics Intact Mass software was used for rawdata deconvolution. Thermo Xcalibur Qual Browser was used for extractedion chromatogram analysis.

Example 1 1.1 Optimization of the Competitive Binding Assay

A competitive binding assay was developed to differentiate variants of aprotein of interest with impaired binding. mAb1 was used as an exemplaryprotein of interest.

mAb1 antigen was biotinylated using a biotinylation reagent (30 min atroom temperature, using NHS-biotin). Biotinylated mAb1 was loaded onto abed of streptavidin-resin (7 nmol biotin binding capacity, Pierce) in atube (micro BioSpin, Bio-Rad). After five minutes, filtration waseffected by centrifugation, and the gel bed was washed with 100 mM Tris,pH 7.5 (about 1 minute incubation, then spin) and then six times withpurified water (Milli-Q, Millipore), to obtain antigen-immobilizedresin.

A series of tubes with eleven different increasing volumes ofantigen-immobilized resin (1-40 μL) were suspended in binding assaybuffer (binding assay buffer can be anything from Tris to PBS buffer).On addition of purified mAb1, the tubes were incubated for 1 hour at 4°C. On centrifugation i.e., spinning down at 800×g for five minutes at 4°C., the supernatant was removed and the binding was analyzed bymeasuring the protein concentration of the flowthrough at 280 nm using aNanoDrop UV-Vis spectrophotometer, relative to the total amount of mAb1.An exemplary embodiment of a titration curve obtained is shown in FIG. 6. The volume of antigen-immobilized resin was insufficient to captureall of the mAb1 sample, thus providing a flowthrough enriched for anybinding-impaired variants of mAb1. The resin volume needed to obtain 50%binding of mAb1 was selected for further competitive binding assays.

1.2 Competitive Binding-nSCX-UV/MS Analysis.

A competitive binding assay as described above was performed for mAb1.

SCX-UV analysis of mAb1 shows that it features a substantial glycationvariant, as shown in FIG. 7 . The specific glycation site was identifiedas heavy chain (HC) lysine (K) 98, as shown in Table 1. This glycationhas previously been implicated in antigen-binding, but its exact impactwas unknown.

TABLE 1 PTM Site Peptide Percentage PTM Location^(a) Sequence (%)Asparagine HC Asn⁸⁴ (K)NSLFLQMNSL 1.1% R(A) Deamidation HC Asn³¹¹/(R)VVSVLTVLHQ 2.5% HC* Asn³¹⁸ DWLNGK(E) HC Asn³⁸⁰/ (K)GFYPSDIAVE 4.0%HC*Asn³⁸⁷ WESNGQPENNYK(T) Methionine HC Met²⁴⁸/ (K)DTLMISR(T) 3.7%Oxidation HC* Met2⁵⁵ HC Met⁴²⁴ (R)WQEGNVFSCSV 1.2% MHEALHNHYTQK(S)HC* Met⁴³¹ (R)WQEGNVFSCSV 1.4% MHEALHNR(F) Lysine HC Lys⁹⁸ (E)DTAVYFCV31.7% Glycation K(D) Lysine 4.3% Glucuronylation Lysine 1.0% Carboxy-methylation Lysine +161.01 1.3% Da Modification

In order to determine whether any major variants of mAb1, such asglycation, impact antigen-binding, mAb1 flow through from thecompetitive binding assay was compared to a control sample of mAb1 usingSCX-UV analysis. The control experiment included use of SCX-UV/MS on themAb1 obtained from stability study without any enrichment step. Thecomparison of the two chromatograms is shown in FIG. 8 . FIG. 8 clearlyshows enrichment of the glycation peak of mAb1 in flow through from thecompetitive binding assay as compared to control mAb1. This demonstratesthat the glycation modification indeed impairs binding of mAb1 to mAb1antigen, and is therefore a CQA that should be taken into considerationin product development.

1.3 Evaluation of Multiple Critical Quality Attributes Using CompetitiveBinding-SCX-MS.

The samples from Example 1.2 were further subject to mass spectrometryanalysis. Extracted-ion chromatograms (XIC) from the mAb1 control sampleand the mAb1 flow through of the competitive binding assay are shown inFIG. 9 . Several protein variants are identifiable in the compared XICs,demonstrating that the method of the invention is capable ofsimultaneously identifying several CQAs that adversely affect proteinbinding. At the same time, PTMs that have no change in relativeabundance between the samples may be disregarded as CQAs.

A statistical analysis of the enrichment of PTMs in the competitivebinding assay flow through compared to the control sample is shown inFIG. 10 . It is clear from the comparison that some modifications (suchas HC K98 glycation, HC K98 carboxymethylation (CML), and HC K98glucuronylation) were enriched using the competitive binding assayexperiment, identifying them as CQAs for mAb1, while others (N-term Qand terminal galactosylation of Fc glycan) were not. Thus, the methodsuccessfully identified critical quality attributes and product-relatedvariants that cause reduced binding of mAb1 to mAb1 antigen, anddistinguished them from modifications that do not impact binding andthus can be disregarded in product development.

Example 2 2.1 Competitive Binding-SCX-MS Analysis of a BispecificAntibody

The effectiveness of the method of the invention was furtherdemonstrated by analysis of a bispecific antibody, bsAb1. The structureof bsAb1 is shown in FIG. 11 . FIG. 11 shows that mAb2 comprises twodistinct HC regions, HC and HC*.

Previous nSCX-MS analysis of bsAb1 lots has shown that it prominentlyfeatures a deamidation variant. Previous peptide mapping analysis hasidentified a major variant caused by deamidation at HC N56, as shown inTable 2.

TABLE 2 t0 in Platform, pH6, Site Peptide Platform 45C28d Loca- Se- PTMMod Total Mod Total tion quence name (%) (%) (%) (%) HC Q VQLVE Q1 >94.2 94.2 99.6 99.6 Gln1 SGGGWQ pyro- PGR Glu HC LSCAAS M34 0.2 0.2 0.20.2 Met34 GFTFSS Oxida- YG M HWV tion R HC GLEWVA N56 15.1 15.1 30.430.4 Asn56 VISYAG Deamida- N NK tion HC DSYYDF D99 0.2 0.2 1.9 1.9 Asp99LTDPDV Dehydra- LDIWGQ tion GTMVTV SSASTK HC DSYYDF M119 3.4 3.4 9.3 9.3Met119 DPLTDV Oxida- GLDIWQ tion GT M VTV SSASTK HC DTL M IS M255 5.25.2 8.7 8.7 Met255/ R Oxida- HC* tion Met255

HC N56 is located in the complementarity-determining region (CDR) ofbsAb1, raising the possibility that it may adversely impact binding ofbsAb1 to its target. In order to determine any potential impact of bsAb1variants on binding, bsAb1 was subjected to competitive binding-SCX-MSanalysis.

Antigen-immobilized resin was optimized and prepared as described inExample 1.1. bsAb1 was subjected to the competitive binding assay, andflow through from the competitive binding assay was compared to bsAb1control sample using SCX-UV/MS. The comparison of the two UVchromatograms is shown in FIG. 12 . FIG. 12 clearly shows enrichment ofa deamidation variant of bsAb1 in the flow through from the competitivebinding assay, which is quantified as shown in FIG. 13 . The enrichmentof a deamidation variant of bsAb1 in the flow through of the competitivebinding assay demonstrates that deamidation is a CQA in the productionof bsAb1.

2.2 Evaluation of Multiple Critical Quality Attributes Using CompetitiveBinding-SCX-MS.

bsAb1 was subjected to further analysis using competitivebinding-SCX-peptide mapping MS. The extracted-ion chromatograms (XIC)from the control experiment and the competitive binding assay flowthrough showed several different PTMs. A comparative analysis of theamount of variants obtained using the control experiment and thecompetitive binding assay experiment is shown in FIG. 14 . It is clearfrom the comparison of the variants that only the N56 deamidationvariant was enriched using the competitive binding assay experiment, andthus was probably the only identified critical quality attribute orproduct-related variant of bsAb1 with reduced binding affinity.

What is claimed is:
 1. A method for characterizing at least oneproduct-related variant, said method comprising: a. obtaining a sampleincluding a protein of interest and at least one product-related variantof said protein of interest; b. contacting said sample to a competitivebinding condition including an insufficient target immobilized on beads;c. washing said beads to collect a flow-through; d. subjecting saidflow-through to liquid chromatography-mass spectrometry analysis toseparate said protein of interest and said at least one product-relatedvariant; and e. comparing the abundance of said at least oneproduct-related variant from (d) to an abundance of said at least oneproduct-related variant obtained from a liquid chromatography-massspectrometry analysis of a control sample of (a) to characterize said atleast one product-related variant.
 2. The method of claim 1, wherein theliquid chromatography is strong cation exchange chromatography.
 3. Themethod of claim 1, wherein said beads are agarose beads or magneticbeads.
 4. The method of claim 1, wherein said flow-through is enrichedfor said at least one product-related variant.
 5. The method of claim 1,wherein said flow-through of (c) is collected by performingcentrifugation.
 6. The method of claim 1, further comprising subjectingsaid flow-through of (c) to digestion conditions prior to liquidchromatography-mass spectrometry analysis.
 7. The method of claim 1,wherein said beads are coated with streptavidin resin.
 8. The method ofclaim 1, wherein said insufficient target includes an amount of saidtarget capable of binding to about 30% to about 80% of said protein ofinterest.
 9. The method of claim 1, wherein said sample of (b) isincubated for about one hour.
 10. The method of claim 1, wherein saidsample of (b) is incubated at about room temperature.
 11. The method ofclaim 1, wherein said product-related variant comprises a size-variant.12. The method of claim 11, wherein said size-variant is a fragmentationvariant of said protein of interest.
 13. The method of claim 11, whereinsaid size-variant is an aggregation variant of said protein of interest.14. The method of claim 1, wherein said product-related variantcomprises a charge-variant of said protein of interest.
 15. The methodof claim 1, wherein said product-related variant comprises a posttranslationally modified-variant of said protein of interest.
 16. Themethod of claim 1, wherein said target is an antigen directed to saidprotein of interest.
 17. The method of claim 1, wherein saidproduct-related variant is characterized as a critical quality attributeif said abundance of said at least one product-related variant from (d)is significantly more than said abundance of said at least oneproduct-related variant obtained from the liquid chromatography-massspectrometry analysis of the control sample of (a).